Therapeutic agents

ABSTRACT

wherein R is selected from the group consisting of elaidoyl, cis-eicosenoyl and trans-eicosenoyl; and pharmaceutical compositions comprising the Ara-C derivative.

This Application is both (a) a divisional of U.S. patent applicationSer. No. 08/983,483, which was PCT filed on May 28, 1998, and (b) aContinuation-In-Part of U.S. patent application Ser. No. 08/532,754,which was PCT filed on Apr. 5, 1994, and which issued as U.S. Pat. No.6,153,594 on Nov. 28, 2000.

This invention relates to certain nucleoside derivatives which have beenfound to have valuable properties for the treatment of tumours.

The nucleoside derivatives are esters of 1-β-D-arabinofuranosylcytosine(Ara-C) of formula A:

Ara-C is also sometimes known as cytosar.

Ara-C has long been known as a chemotherapeutic agent in the treatmentof acute myelogenous leukaemia but has limited efficiency against solidtumours (Fre et al., Cancer Res. 29 (1969), 1325-1332; Davis et al.,Oncology, 29 (1974), 190-200; Cullinan et al., Cancer Treat. Rep. 61(1977), 1725-1726). However, even in the treatment of leukaemia Ara-Chas found only limited use due to its very short biological half-lifeand its high toxicity.

With a view to overcoming these difficulties, a number of workers haveprepared and tested pro-drug derivatives of Ara-C. For example, Hamamuraet al. investigated 3′-acyl and 3′,5′-diacyl derivatives of Ara-C (J.Med. Chem. 19 (1976) No. 5, 667-674). These workers prepared and testednumerous Ara-C derivatives with saturated or unsaturated ester groupscontaining from 2 to 22 carbon atoms, and they found that many of thecompounds showed a higher activity against L1210 Leukaemia in mice thanthe parent nucleoside alone.

The work by Hamamura et al., and others, on pro-drug analogues of Ara-Cwas reviewed by Hadfield et al. in Advances in Pharmacology andChemotherapy, 20, 1984, pages 21-67. In discussing 5′-esters of Ara-C,these authors conclude (page 27):

“ . . . though many of these agents appear to function as very efficientdepot forms of ara-C in mice, the analogous action in man has not beendemonstrated.”.

Although work has continued on pro-drugs based on Ara-C, including 3′-and 5′-acyl derivatives (see, for instance, Rubas et al. in Int. J.Cancer, 37, 1986, pages 149-154 who tested liposomal formulations of,inter alia, 5′-oleyl-Ara-C against L1210 Leukaemia and Melanoma B16) todate no such drugs have become available to the clinician.

The mode of action of Ara-C relies on its enzymatic recognition as a2′-deoxy-riboside and subsequent phosphorylation to a nucleosidetriphosphate which competes with the normal CTP for incorporation intoDNA. The 2′-hydroxyl group causes steric hindrance to rotation of thepyrimidine base around the nucleosidic bond. The bases ofpolyarabinonucleotides cannot stack normally, as do the bases ofpolydeoxynucleotides. Ara-C inhibits DNA repair and DNA synthesis bothby slowing down chain elongation and movement of newly replicated DNAthrough the matrix-bound replication apparatus. The mechanism of actionof Ara-C results in an “unbalanced growth” in dividing cells. Ara-C actsin the S-phase of the cell cycle. For continuos inhibition of the DNAsynthesis and finally cell death, it is crucial that Ara-C is a presentat a sufficiently high concentration during at least one cell cycle.

A main reason why Ara-C is not used in the treatment of solid tumours isagain the rapid clearance of the active drug from cancer cells andplasma. It is apparently not possible to achieve significantintracellular levels of drug in the neoplastic tissue, even though thetumour in question is sensitive to Ara-C in-vitro. The surprisinglyprolonged half life and altered tissue distribution. of the products ofthis invention will be of great importance for the therapeutic effect ofthese products.

We have found, as shown in FIGS. 7, 8 and 9, that 3′- and 5′-O-esters ofAra-C and certain saturated and unsaturated fatty acids unexpectedlyexhibit good activity against different tumours in contrast to Ara-Citself and also other mono- and diesters.

It is felt by the present inventors that the test model which iscommonly used (injection of leukaemia cells into the abdominal cavity ofmice and treated i.p.) is more comparable to an in vitro model than toan actual clinical situation and may have served to hide theparticularly valuable properties of the selected Arab esters used in thepresent invention, as will be described below.

More specifically, the 3′- and 5′-O-esters which are used according tothe present invention are those which are derived from C₁₈ or C₂₀saturated and monounsaturated fatty acids.

Thus, the esters used according to the present invention may berepresented by the formula 1:

wherein R₁ and R₂ are independently selected from hydrogen, and C₁₈- andC₂₀- saturated and mono-unsaturated acyl groups, with the provisos thatR₁ and R₂ cannot both be hydrogen.

The double bond of the mono-unsaturated acyl groups may be in either thecis or the trans configuration, although the therapeutic effect maydiffer depending on which configuration is used.

The position of the double bond in the mono-unsaturated acyl groups alsoseems to affect the activity. Currently, we prefer to use esters havingtheir unsaturation in the ω-9 position. (In the ω-system ofnomenclature, the position (ω) of the double bond of a monounsaturatedfatty acid is counted from the terminal methyl group, so that, forexample, eicosenoic acid (C₂₀:1 ω-9) has 20 carbon atoms in the chainand the single double bond is formed between carbon atoms 9 and 10counting from the methyl end of the chain). Thus, we prefer to use Ara-Cesters derived from oleic acid (C₁₈:1, ω-9, cis) elaidic acid (C₁₈:1,ω-9, trans) and eicosenoic acid (C₂₀:1, ω-9, cis) and (C₂₀:1, ω-9,trans) and stearic acid (C₁₈:0) and eicosanoic acid (C₂₀:0).

Both 3′-O- and 5′-O-monoesters and 3′, 5′-O-diesters can be used in thetreatment of different tumours in accordance with the present invention,but in general the 5′-O-monoesters are preferred. The 3′,5′-O-diestersare expected to be useful in those cases where lipophilic properties areof advantage, e.g. absorption or uptake in lipid tissues.

The compounds of formula (I) wherein R₁ and R₂ are independentlyselected from hydrogen, elaidoyl, oleoyl. stearoyl, eicosenoyl (cis ortrans) and eicosanoyl, with the provisos that R₁ and R₂ cannot both behydrogen, oleoyl or stearoyl, R₁ cannot be hydrogen when R₂ is oleoyl orstearoyl, and R₂ cannot be hydrogen when R₁ is elaidoyl, oleoyl orstearoyl, are new compounds not previously reported in the prior art.

More specifically these new compounds of formula (1) are defined in thebelow Table A wherein R₁ and R₂ are as given:

TABLE A R₁ R₂ hydrogen elaidoyl hydrogen eicosenoyl (cis) hydrogeneicosenoyl (trans) eicosenoyl (cis) hydrogen eicosenoyl (trans) hydrogeneicosenoyl (cis) eicosenoyl (cis) eicosenoyl (trans) eicosenoyl (trans)eicosenoyl (cis) eicosenoyl (trans) eicosenoyl (trans) eicosenoyl (cis)eicosenoyl (cis) elaidoyl eicosenoyl (trans) elaidoyl elaidoyleicosenoyl (cis) eiaidoyl eicosenoyl (trans) eicosenoyl (cis) oleoyleicosenoyl (trans) oleoyl oleoyl eicosenoyl (cis) oleoyl eicosenoyl(cis) eicosanoyl eicosanoyl eicosanoyl stearoyl stearoyl eicosanoylelaidoyl stearoyl eicosenoyl (cis) stearoyl eicosenoyl (trans) stearoylelaidoyl eicosanoyl eicosenoyl (cis) eicosanoyl eicosenoyl (trans)eicosanoyl stearoyl oleoyl oleoyl stearoyl

A limiting factor for the use of Ara-C is its degradation by cytidinedeaminase and deoxycytidine-monophosphate (dCMP) deaminase to inactivemetabolites. We have surprisingly found that the monoesters of thisinvention are poor substrates for these deactivating enzymes. Thisdifference could imply that these ester-derivatives are more suited thanAra-C itself for systemic or local treatment of malignant tumours,especially malignant tumours in the RES and CNS.

This is clearly demonstrated in the leukaemia brain-metastasis modeldescribed in FIGS. 10, 11 and 12 and especially with the more aggressiveB-cell lymphoma shown in FIG. 11 were Ara-C itself is void of activity.

In the clinical treatment of myelogenous leukaemia, the rapiddeactivation of Ara-C is compensated by continues infusion over 5-7 daysto establish a reasonably stable therapeutic active plasma level ofAra-C. We have shown that following intravenous administration ofequimolare amounts of radio labelled Ara-C and Ara-C-5′-elaidic ester torats, a beneficial change in the metabolism rate and excretion profileis achieved. As can be seen from table 1 and FIG. 20, the administrationof Ara-C-5′-elaidic ester gives both a higher initial whole blood andplasma concentration and a slower conversion to Ara-U. The deaminationto Ara-U from Ara-C of the esters of this invention, here exemplified byadministration as the elaidate is observed as significantly slower, andwhen the plasma levels of both Ara-C and Ara-U is below the assay limitof detection at 48 h when administered as pure Ara-C, the two compoundscan still be quantified at 72 h following administration ofAra-C-5′-elaidate. As can be seen from table 2, the total excretedamount of Ara-U (AUC, 072 h) is the same for both administeredcompounds. In a clinical situation these results are reflected in abroader time window of therapeutic active concentration of Ara-C in theblood. In the in-vivo leukaemia model described in FIG. 13, Ara-C andthe 5′-elaidic ester are compared, and similar anti-cancer effectsachieved with Ara-C are demonstrated with administration of {fraction(1/20)} of the molar dose of the ester.

If a similar toxicity profile that is seen in the dinic with Ara-C isobserved with the ester derivatives, the improvement in therapeuticindex should be of the same order (×20) of magnitude as the dose/effectimprovement.

Of major importance in the treatment of leukaemia's and other diseasesconfined to the reticule endothelial system (RES) is of course the timewindow of active drug plasma concentration, but a localisation of theactive compound in RES tissues (defined as liver, spleen, lymph, lung,intestine wall and free phagocytotic cells present in for example. bonemarrow and whole blood) will be of great importance as well. We haveobserved (FIG. 21 and table 1) that by intravenous administration ofequimolar amounts of Ara-C and Ara-C-5′-elaidic ester, the concentrationof active drug in RES tissues is significantly higher, and persists witha broader time window when dosing the ester derivative. The pattern ofdistribution and metabolism is investigated in greater detail andresults from the liver is given in FIG. 19. A therapeutic significantAra-C level is sustained for at least 72 h after dosing the esterderivative. This can enable treatment of original liver cancer or livermetastases of colo-rectal, breast, melanoma or other forms of cancer.The treatment can stand as mono-therapy, or as paliative/adjuvanttreatment in combination with surgery, radiation or other chemotherapy.

There is also observed increased concentrations of Ara-C in othertissues, and this combined with a smaller volume of distribution mayopen for therapy with Ara-C esters in cancer forms not normallyassociated with Ara-C treatment.

Moreover we unexpectedly found that the esters of this invention (FIG.15) stimulated to a large degree the activation of NFkappaB whilst Ara-Cgave no stimulation. The stimulation is a biological effect not normallyseen with therapeutic chemicals, and in particular not with conventionalcytostatics. This could suggest that the Ara-C esters of this inventionhave a stimulating effect on certain immune factors which again couldexplain the astonishing improvement in anti-cancer effect. This could beof significant importance in the treatment of neoplastic diseasesinvolving immunocompetent cells such as leukaemia's and lymphomas.

The development of resistant cancer cells is a severe problem in thecurrent chemotherapy of cancer. We have found (FIGS. 7-9) that the Ara-Cderivatives of this invention show the same effect against Cis-platinresistant cells (NHIK 3025/DDP) and MDR resistant cells (A549) asagainst the corresponding non resistant cell lines. This, we believe, isbecause the derivatives are not substrates for the cellular drug-efluxmechanisms, such as the “gp 120 MDR pump”, responsible for thephenomenon seen as multi drug resistance.

The C₁₈ and C₂₀ mono- and di-esters of Ara-C can be used according tothe present invention in the treatment of a number of neoplastictumours. We have found an especially promising effect on brain tumourssuch as glioma, and metastasis from other tumours such as sarcomas,carcinomas. as well as leukaemia. Currently, glioma are treated bysurgery, radiation therapy and cytostatica, e.g.N,N-cis(2-chloroethyl)N-nitroso-urea (BCNU). However, the prognosis bythese treatments is very poor.

Useful effects with the Ara-C esters of the present invention have alsobeen found in metastatic tumours, such as carcinoma, sarcomas, leukaemiaand melanomas.

The scope of the invention and its essential and preferred features areas defined in the attached claims.

Biological Effects

Micellar Formulation

A 1 mg/ml micellar formulation is prepared by the 1:1 (w/w) mixing ofAra-C ester (in DMSO) and lecithin (in ethanol) in sterile water.

Clonogenic Agarose Assay¹

A biopsy was taken from the patient and placed immediately in a growthmedium. Tumour tissue was desegregated mechanically, and living cellswere selected the chemotherapeutic test substance was added, BCNU (inwater) and Ara-C and Ara-C esters (in micelles), and the cells werecultivated in a soft agarose medium. Twenty-four hours beforetermination of the cultures (7 days) ³H Thymidin was added. The activityof the test substance is thus quantified as cpm in a scintillationcounter.

⁽¹⁾ G. Unsgaard et al., Acta Neurochir (Wien) (1988) 91:60-66.

FIG. 1

The results here are obtained with a glioblastoma taken from a patient.The same response pattern is found in 8 other glioblastoma biopsies. Thegraph shows the in vitro comparison of Ara-C and its 3′-elaidyl esterand 5′-elaidyl ester. The results are given as % of the untreatedcontrol. A count of 50% (CD₅₀) is taken as promising regarding use intherapy of this actual cancer line. What is worth nothing here is the10^(1.5) higher concentration of Ara-C needed to obtain CD₅₀ as comparedwith elaidyl esters.

FIG. 2

Shows the results obtained with the same glioblastoma as FIG. 1. Thegraph compares radiation therapy and chemotherapy (BCNU). A radiationdose greater than 10 Gray (Gy) needed to obtain CD₅₀ is in no sensepractical in therapy. Comparing FIGS. 1 and 2, the concentration neededof BCNU to obtain CD₅₀ is about 10 times higher than what is needed withthe Ara-C esters, but reasonably comparable to Ara-C alone.

FIG. 3

These results are obtained with a biopsy taken from a brain metastasisof a melanoma. The difference here between Ara-C and the 3′- and5′-elaidyl esters is not as pronounced as with the glioma, but is stillof the order of 10 times higher.

FIG. 4

This shows the activity of BCNU on the melanoma cell line. Compared tothe Ara-C esters the BCNU is here needed in more than 1×10² higherconcentration to give CD₅₀.

FIG. 5

This graph shows results with brain metastasis of a carcinoma (lung).These cancer cells are more resistant to chemotherapy, but thedifference between Ara-C and Ara-C esters are still present.

FIG. 6

These results with BCNU treatment of brain metastasis of a carcinoma(lung) which are presented here are similar to what has already beendemonstrated with the other cell lines.

Regarding the different cell types investigated, there seems to be anexplicit difference in activity between the Ara-C esters, Ara-C aloneand BCNU. A potentiation of 1×10² is very promising for a therapysituation. The findings indicate that the 5′ esters are somewhat morepotent than the 3′ esters.

Cell Inactivation—Colony Forming Ability

Cell inactivation measured by means of loss of ability to form colonieswas determined for several compounds. Cells used were of the establishedhuman cell lines of cancer cervix in situ origin, NHIK 3025, NHIK3025/DDP, a cis-DDP-resistant variant of the same or A549 cells (humanlung carcinoma). The cells were exposed to the test compound for 4 uptill 24 hours. Test compounds were administered as micellar solution.Number of colonies were counted after about 12 days of incubation.

FIG. 7

The graph shows the in vitro comparison of the test compounds Ara-C,Ara-C-5′-elaidyl ester, Ara-C-5′-stearyl ester, Ara-C-5′-eicosen esterand Ara-C-5′-petroseline ester. The results are given as the dose neededto reduce cell survival with 90% relative to untreated control. As seenfrom the graph, substantial higher inactivation of NHIK 3025 cells isobserved following exposure to the esters compared to Ara-C itself. Thedose modifying factor at the 10% survival level is in the range of 3 to5 for the Ara-C-esters compared to Ara-C, which means that a 3 to 5times higher dose is required of Ara-C to obtain similar reduced colonyforming ability as that observed for the esters.

FIG. 8

The results here are obtained with 4 h treatment of NHIK 3025/DDP cells.Enhanced effect of Ara-C-5′-elaidate ester compared to effect of Ara-Cis observed similar to the effect enhancement observed in NHIK 3025cells. Enhanced effect does not depend on resistance to cis-DDP.

FIG. 9

The graph shows the in vitro results using A549 cells (human lungcarcinoma cells) colony forming ability to compare the test compoundsAra-C, Ara-C-5′-elaidyl ester, Ara-C-5′-stearyl ester, Ara-C-5′-eicosenester and Ara-C-5′-petroseline ester. The cells were exposed for 24hours. The highest inactivation is observed for Ara-C-5′-stearyl ester,but enhanced effect is also observed for the elaidyl and petroselineesters.

Raii Human B-lymphoma Cells—Leptomeninpal Carcinomatosis Model in NudeRats

The model used is a tumour model in nude rats for leptomeningal growthof tumours. 1×10⁶ cells of the Bcell tumour line Raji were injected intothe spinal fluid through cistema magna (c.m.). of 4-5 weeks old nuderats. The animals develop neurological symptoms after 12-14 days ifuntreated. Anaesthetised animals were treated intracerebrally with a 40μl injection into cistema magna with 3 or 4 bolus injections. Treatmentwas started 1 day after cell inoculation. Test compounds wereAra-C-5′-elaidyl ester (in micelles) and Ara-C. Ara-C was administeredboth at maximal tolerable dose (MTD) and at an equimolar dose toAra-C-5′-elaidyl ester. Control animals (treated with NaCl) or emptyliposomes (micelles without Ara-C esters) developed symptoms from thecentral nervous system after approximately 14 days.

FIG. 10

3 bolus injections with Ara-C-elaidate on day 1,2 and 4 increased thesymptom free latency period by 135% as compared to Ara-C, with mean dayof death delayed from day 13 till day 30.5, as seen in FIG. 10. One ratsurvived for more than 70 days, and was considered to be cured. Notumours were visible at necropsy on day 76. This increase indisease-free survival is superior to results obtained with othertherapeutic alternatives tested in comparable models for different typesof human tumours.

FIG. 11

Survival curves from an additional experiment with nude rats inoculatedwith Raji cells in the brain, treated with 4 bolus doses is shown inthis figure. One daily bolus dose on day 1,2,3 and 4 were administeredinto cistema magna. As in the previous experiment, no effects wereobserved for Ara-C, neither at maximal tolerable dose of Ara-C (MTD) norat a dose equimolar to Ara-C-elaidate. The results for the group givenAra-C-elaidate were even more astonishing than in the previousexperiment 3 out of 5 rats were still alive and symptom-free at day 70.They were considered to be cured. This is most promising 5/6 controlrats died on day 13. The 6th control rat had no backflow of spinal fluidinto the syringe following injection of tumour cells and no neurologicalsymptoms after 70 days. According to normal procedure this animal isleft out of the results.

Molt 4 Human Lymphoma Cells—Leptomeningal Carcinomatosis Model in NudeRats

The model used is a tumour model in nude rats for leptomeningal growthof tumours. 10⁶ cells of the T-cell tumour line Molt 4 were injectedinto the spinal fluid through cistema magna (c.m.) of 4-5 weeks old nuderats. The animals develop neurological symptoms after 20-22 days ifuntreated. Anaesthetised animals were treated intracerebrally with a 40μl injection into cistema magna with 4 bolus injections. Treatment wasstarted 1 day after cell inoculation. Test compounds wereAra-C-5′-elaidyl ester (in micelles) and Ara-C. Ara-C was administeredboth at maximal tolerable dose (MTD) and at an equimotar dose toAra-C-5′-elaidyl ester. Control animals (treated with NaCl) developedsymptoms from the central nervous system after approximately 20 days.

FIG. 12

Survival as a function of time for rats injected in the brain with Molt4 lymphoma cells, treated 4× in cistema magna is shown in FIG. 12. Inthis initial experiment, onset of death was delayed for the animalsreceiving Ara-C-elaidate compared to animals receiving Ara-C or control.The number of animals per group were: Control (7), Ara-C-elaidate (3)and Ara-C (5).

Leukaemia Model Using Raii Human B-lymphoma Cells

SCID mice were injected intravenously with 1×10⁶ Raji human B-lymphomacells. The mice were treated on days 7, 9, 11, 13 and 15 followinginjection of the tumour cells with either 20 mg/kg/day of Ara-C-elaidateor 200 mglkg/day of Ara-C or control. Animals develop paralysis of thehind legs as a result of the tumour growth. Mean day of death for theanimals treated with the different treatments are shown in FIG. 13.

FIG. 13

Mean survival of SCID mice injected with Raii human B-lymphoma cellsintravenously, treated intravenously with one injection on each of thedays 7,9,11,13 and 15 with either Ara-C-elaidate, Ara-C or Control isshown in this figure. The doses were 20 mg/kg of Ara-C-elaidate and 200mg/kg for Ara-C. On an equimolar basis, a 20 times reduced dose ofAra-C-elaidate compared to the Ara-C dose increased mean survivalcompared to control and Ara-C treated animals. The number of animals ineach group were 7.

FIG. 14

Mean survival of SCID mice injected with Raji human B-lymphoma cellsintravenously, treated intraperitoneally with once daily injection days7-11 with either Ara-C-elaidate, Ara-C or Control is shown in thisfigure. The mean survival time is greatly prolonged for theAra-C-elaidate when treatment is repeated daily instead of every otherday.

Activation of the Cellular Transcription Factor NFkappaB

Human SW480 colon adenocarcinoma cells stably transfected with a CMVpromotor/enhancer containing the gene for β-galactosidase were used.Activation of the transcription factor NFkappaB results in enhancedamount of the enzyme β-galactosidase in cytoplasma. The amount ofβ-galactosidase is quantified using optical density at 570 nm asparameter. The SW380 cells were incubated 2-3 days before exposure tothe test compound for 4 h. The cells were washed and prepared, andoptical density recorded for the different compounds.

FIG. 15

No β-galactosidase activity was measured following exposure to Ara-C,whilst a substantial increase in β-galactosidase activity was observedas an increase in optical density at 570 nm following exposure toAra-C-elaidate. This indicates that a surprisingly high induction of thetranscriptional activator protein NFkappaB is obtained withAra-C-elaidate. NFkappaB is involved in gene control of a range ofimmune factors, and this activation by Ara-C-elaidate could explain theimproved anticancer effects observed for Ara-Celaidate. One would expecta stimulation of certain immune cells by Ara-C-elaidate, which could beof special interest in the treatment of leukaemia and lymphomas.

The anti-tumour Activity of Ara-C-elaidate Versus Ara-C Against theMurine TLX/5 Lymphoma.

CBA mice weighing 20-25 g were inoculated subcutaneously inguinally with1×10⁵, TLX/5 tumour cells day 0. Ara-C-elaidate or Ara-C wereadministered intraperitoneally on days 3,4,5,6 and 7. Doses were in therange 6.25-50 mg/kg/day. There were 5 mice per treatment per group and10 tumour-bearing controls. Activity was assessed in terms of increasein life span (ILS) versus controls.

FIG. 16

TLX/5 lymphoma tumour bearing mice and their % increase in lifespan(median of 5 per group) following treatment with Araelaidate or Ara-C,i.p treatment for 5 days is shown in this figure. Ara-C was only activeat the dose 25 mg/kg, whilst Ara-C-elaidate was active at the doses 12.5mg/kg and 25 mg/kg. Maximum increase in lifespan was 47.2% compared to32.7% for Ara-C.

The Anti-tumour Activity of Ara-C-elaidate Versus Ara-C in SCID MiceInoculated Intraperitoneally With Hemangiosarcoma Cells.

SCID mice were inoculated intraperitoneally with PV/2b/35hemangiosarcoma cells. Mice were treated 5 days per week with 25mg/kg/day of either Aras-C-elaidate prepared in micelles, Ara-C-elaidatedissolved in DMSO, Ara-C dissolved in PBS. Controls were empty micelles,DMSO or PBS respectively. The animals were not treated during weekends.Survival was the endpoint of the study.

FIG. 17

Survival of SCID mice inoculated intraperitoneally with PV/2b/35hemangiosarcoma cells. Survival was greatly enhanced for animals treatedwith Ara-C-elaidate. The enhanced survival compared to control wasobserved both for Ara-C-elaidate prepared in micelles and forAra-C-elaidate dissolved in DMSO.

FIG. 18

The results presented here are from a study of the 5′-Ara-C elaidylester in a glioblastoma tumour grown in nude mice. A glioblastoma cellline U-118 (Uppsala) tissue culture was injected subcutaneously in nudemice. A small part (2×2 mm) of growing tumour was transferred to newmice. The subcutaneous tumours show a somewhat different growth rate inthe various animals, but at the size of 4-6 mm, an injection with a 10mg/ml micellar solution of the Ara-C ester was given intratumourally.Depending on the actual tumour size, the animals received the samerelative amount of test substance. The control was given saline water.The growth rate was recorded as relative tumour volume (RTV). Thecontrol tumour follows a quite normal growth pattern typical to thiscancer type. What is noted is the complete stop in tumour growth of thetreated animals. Further, the animals showed no signs of toxic sideeffects, which in the case of Ara-C are damage to bone marrow with thedevelopment of anaemia or haemorrhages, nor was there noted any sign ofCNS disturbance.

Comparative Pharmacokinetic, Distribution, Metabolism and Excretion of¹⁴C-Ara-C-elaidate and ¹⁴C-Ara-C Administered Intravenously to Male Rats¹⁴C-Ara-C-elaidate (in micelles) or ¹⁴C-Ara-C were administeredintravenously to male rats at equimolar doses, 5 mg/kg for¹⁴C-Ara-C-elaidate and 2.4 mg/kg for ¹⁴C-Ara-C. Plasma concentrations oftotal radioactivity and of the metabolites were determined at differenttimepoints. Tissue concentrations of total radioactivity were determinedfrom a range of tissues at different timepoints up to 120 hoursfollowing injection. Liver tissues were extracted and metaboliteconcentrations were determined up to 72 hours post injection. Tissuedistribution of Ara-C-elaidate was significantly altered compared to thedistribution of Ara-C. Maximal concentrations in most tissues werenotably higher and occurred at later timepoints following¹⁴C-Ara-C-elaidate administration, especially in whole-blood/plasma,spleen, liver and lungs. Maximal concentrations in muscle, salivaryglands, skin and urinary bladder were lower. The proportion of the dosein whole-blood at 0.08 hours after ¹⁴C-Ara-C-elaidate administration wasestimated to be 64.7%, notably higher than the proportion present in thesystemic circulation at this time after ¹⁴C-Ara-C administration(7.76%). Excretion via the renal system was much slower for the elaidatethan the Ara-C itself. Elimination from tissues were much slower for¹⁴C-Ara-C-elaidate compared to elimination from tissues when ¹⁴C-Ara-Cwas administered.

Table 1

Maximal concentrations of radioactivity (expressed as μg equivalents/g)following administration of equimolar doses of ¹⁴C-Ara-C-eiaidate or¹⁴C-Ara-C with corresponding timepoint for maximal concentration. Asseen in the table, maximal concentrations occurred in different tissuesand at different timepoints for the two compounds.

TABLE 1 Tissue ¹⁴C-Ara-C-elaidate (t_(max.) hours) ¹⁴C-Ara-C (t_(max.)hours) spleen 175.2 (0.25) 2.406 (0.08) plasma 55.60 (0.08) 3.058 (0.08)whole-blood 47.32 (0.08) 2.707 (0.08) liver 42.37 (1 hour) 2.526 (0.08)blood cells 34.37 (0.08) 2.201 (0.08) lung 28.97 (0.08) 2.144 (0.08)vena cava 17.29 (0.08) 1.887 (0.25) bone marrow 13.29 (1 hour) 1.950(0.08) heart 10.15 (0.08) 1.916 (0.25) kidney 9.108 (0.08) 7.752 (0.08)prostate 9.014 (4 hours) 2.810 (0.25) pituitary 8.359 (0.08) 0.931(0.08) aorta 7.795 (0.08) 2.213 (0.08) urinary bladder 6.421 (4 hours)13.07 (1 hour) adrenal glands 5.229 (0.08) 1.764 (0.08) salivary glands2.366 (0.25) 2.505 (0.08) lacrimal glands 4.438 (4 hours) 2.460 (0.08)lymph nodes 2.831 (1 hour) 2.222 (0.08) skin 1.793 (0.25) 2.189 (0.08)muscle 1.990 (0.25) 2.158 (0.08) pancreas 2.817 (0.08) 2.148 (0.08)thymus 2.090 (0.25) 2.054 (0.08) brain 1.408 (0.08) 0.233 (1 hour)

Table 2

Excretion of radioactivity (% of dose) after intravenous administrationof ¹⁴C-Ara-C-eladiate (5 mg/kg) or ¹⁴C-Ara-C (2.4 mg/kg) to male rats.Rate of excretion of radioactivity in urine is slower for¹⁴C-Ara-C-eladiate than for ¹⁴C-Ara-C.

TABLE 2 Sample/time (hours) ¹⁴C-Ara-C-elaidate ¹⁴C-Ara-C 0-6 59.1 ± 3.7 85.3 ± 3.1   6-24 34.1 ± 2.5  8.8 ± 1.9 24-48 2.7 ± 0.8 0.5 ± 0.3 48-720.5 ± 0.1  0.2 ± <0.1 72-96 0.2 ± 0.1 0.2 ± 0.1  96-120  0.1 ± <0.1 0.1± 0.1

FIG. 19

Liver concentration of Ara-C-elaidate (P-Ara-C-el) and the metabolitesAra-C (P-Ara-C) and Ara-U (P-Ara-U) are plotted as a function of timefollowing injection of ¹⁴C-Ara-C-elaidate in FIG. 15 as well as theconcentration of Ara-C (Ara-C) and the metabolite Ara-U (Ara-U) as afunction of time following injection of 14C-Ara-C itself. Injection ofAra-C-elaidate gave rise to substantially increased and prolongedexposure of rat-liver to both Ara-C-elaidate and Ara-C. with nodetection of Ara-U up to 24 hours. This was in strong contrast to theliver concentration of Ara-C after administration of Ara-C as such.Liver concentration of Ara-C diminished to non-detectable levels after 4hours, with the metabolite Ara-U present at all timepoints.

FIG. 20

Plasma levels of Ara-Celaidate and the metabolites Ara-C and Ara-Ufollowing intravenous administration of ¹⁴C-Ara-C-elaidate is shown as afunction of time as well as plasma levels of Ara-C and the metaboliteAra-U following intravenous administration of ¹⁴C-Ara-C as a function oftime. Ara-C-elaidate administration give rise to prolonged plasma levelof Ara-C, with detectable levels up to 72 hours in plasma followingadministration compared to 24 hours following Ara-C administration.Metabolism of Ara-C to Ara-U is less extensive, and starts later inanimals which have received Ara-C-elaidate.

FIG. 21

Tissue concentration of total radioactivity is plotted as a function oftime following intravenous administration of either ¹⁴C-Ara-C-elaidate(P) or ¹⁴C-Ara-C. The tissues shown in the graph are liver, spleen,lung, bone and bone marrow. Concentration of radioactivity followinginjection of ¹⁴C-Ara-C-elaidate is higher at all timepoints up to 120hours for all the corresponding tissues.

The Ara-C esters of the present invention may be formulated withconventional carriers and excipients for administration.

As the most promising regime for the treatment of gliomas and othersolid brain tumours, we currenty envisage local deposition of the activecompounds at the site of the tumour to be attacked. For this purpose,the active compounds may preferably be presented as a lecithin micellarformulation. For example, the preferred treatment of brain metastasiswill be by administration of a formulation of the Ara-C ester into thespinal fluid or into the tumour area by means of a dosing pump orsimilar device.

The Ara-C esters of the present invention may also be administratedsystemically, either enterally or parenterally.

For enteral administration, the active compounds of the presentinvention may be presented as, e.g. soft or hard gelatine capsules,tablets, granules, grains or powders, drags, syrups, suspensions orsolutions.

When administrated parenterally, preparations of Ara-C esters asinjection or infusion solutions, suspensions or emulsions are suitable.

The preparation can contain inert or pharmacodynamically activeadditives, as well known to those skilled in the formulation arts. Forinstance, tablets or granulates can contain a series of binding agents,filler materials, emulsifying agents, carrier substances or dilutes.Liquid preparations may be present, for example, in the form of asterile solution. Capsules can contain a filler material or thickeningagent i addition to the active ingredient. Furthermore,flavour-improving additives as well as the substances usually used aspreserving, stabilising, moisture-retaining and emulsifying agents,salts for varying the osmotic pressure, buffers and other additives mayalso be present.

The dosage in which the preparations according to this invention areadministered will vary according to the mode of use and route of use, aswell as to the requirements of the patient. In general a daily dosagefor a systemic therapy for an adult average patient will be about0.1-150 mg/kg body weight/day, preferably 1-50 mg/kg/day. For topicaladministration, an ointment, for instance, can contain from 0.1-10% byweight of the pharmaceutical formulation. especially 0.5-5% by weight.

I desired the pharmaceutical preparation containing the Ara-C esters cancontain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine,butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

Combination therapies, i.e. in which the administration of an Ara-Cester of this invention is carried out in conjunction with othertherapies, e.g. surgery, radiation treatment and chemotherapy, are alsocontemplated. For example, the preferred treatment of brain tumoursseems likely to be a combination of surgery and treatment with an Ara-Cester of this invention by systemic or local administration.

The esters of Ara-C used according to the invention may generally beprepared according to the following reaction equation:${{Nu}{—OH}} + {{FaX}\quad \overset{Base}{\underset{- {HX}}{\rightarrow}}\quad {{Nu}{—O—Fa}}}$

wherein Nu-OH stands for Ara-C. O is oxygen at 3′ and for 5′ position ofthe sugar moiety of Ara-C, Fa is an acyl group of a saturated ormonounsaturated C₁₈ or C₂₀ fatty acid, and X may be Cl, Br or OR′wherein R′ is Fa, COCH₃, COEt or COCF₃.

Thus, the reaction proceeds by acylation of the nucdeoside. This isaccomplished by the use of suitable reactive derivatives of fatty acids,especially acid halides or acid anhydrides. When an acid halide such asan acid chloride is used, a tertiary amine catalyst, such astriethylamine, N,N-dimethylaniline, pyridine orN,N-dimethylaminopyridine is added to the reaction mixture to bind theliberated hydrohalic acid. The reactions are preferably carried out inan unreactive solvent such as N,N-dimethylformamide or a halogenatedhydrocarbon, such as dichloromethane. If desired any of the abovementioned tertiary amine catalysts may be used as solvent, taking carethat a suitable excess is present The reaction should preferably be keptbetween 5° C. and 250° C. After a period of 24 to 60 hours, the reactionwill be essentially completed. The progress of the reaction can befollowed using thin layer chromatography (TLC) and appropriate solventsystems. When the reaction is completed as determined by TLC, theproduct is extracted with an organic solvent and purified bychromatography and/or recrystallization from an appropriate solventsystem. As more than one hydroxyl group and also an amino group arepresent in Ara-C, a mixture of acylated compounds will be produced. Theindividual mono- and di-O-esters required may be separated by, forinstance, chromatography, crystallisation, supercritical extraction etc.

When it is desired to prepare a diester compound of formula I, in whichR₁ and R₂ are the same acyl group, it is preferred to employ the abovemethod using the appropriate acyl chloride in excess.

In order to prepare a diester compound of formula I, in which R₁ and R₂are different, it is preferred to first prepare either the 3′- or5′-monoester and then react the monoester with the proper acyl chloride.

This will be exemplified by the following working examples.

EXAMPLE 1

5′-O-(Elaidoyl) 1-β-D-arabinofuranosylcytosine^(1.2)

To a suspension of Ara-C HCl (1.007 g, 3.6×10⁻³ mol) in 15 mldimethylacetamide (DMA) was added a solution of Elaidoyl chloride (1.26g, 4.2×10⁻³ mol) in 5 ml DMA, and the mixture was stirred at 30° C. for22 h. The solvent was evaporated at high vacuum and the residue wastreated with hot ethyl acetate and filtered. The crude product wastreated with 2 M NaHCO₃ aq., filtered off and purified on a column ofsilica gel with methanol (5 30%) in chloroform as the eluent system.Homogenous fractions were recrystallized to give 1.31 g (72%) of thetitle compound as a white solid (mp. 133-134° C.).

¹H NMR (DMSO-d₆, 300 MHz) δ: 7.58(1H, d, H-6), 7.18(2H, br.d, NH₂),6.20(1H, d, H-5), 5.77(1H, d, H-1′), 5.65(2H, m, OH-2′ and OH-3′),5.47(2H, m, CH═CH), 4.43(1H, m, H-5′₁), 4.30(1H, m, H-5′₂), 4.1-4.0(3H,m, H-2′, H-3′ and H4′), 2.45(2H, t, CH₂—COO), 2.05(4H, m, CH₂—C═),1.63(2H, m, CH₂—C—COO), 1.35(20H, m, CH₂), 0.97(3H, t, CH₃).

¹³C NMR (DMSO-d₆, 75 MHz) δ: 172.8(COO), 165.59(C4-N), 155.05(C═O 2),142.86(C-6), 130.11(CH═CH), 92.54(C-5), 86.23(C-1′), 81,86(C-4′),76.83(C-3′), 74.35(C-2′), 63.77(C-5′), 33.46, 31.95, 31.30, 29.03,28.97, 28.85, 28.73, 28.52.28.43, 28.36, 24.48 and 22(CH₂), 13.97(CH₃).

EXAMPLE 2

3′-O(Elaidoyl) 1-β-D-arabinofuranosyl-cytosine^(2.3)

A mixture of 2-hydroxyisobutyric acid (1.15 g, 12×10⁻³ mol) and elaidoylchloride (3. 1 g, 10×10⁻³ mol) was stirred at 50° C. for 1 h. Thionylchloride (1.5 ml, 21×10⁻³ mol was added and stirring was continued for 2h. The reaction mixture was kept by 50° C. at reduced pressure (40 mmHg)for 14 h. The formed 2-elaidoyloxy-2-methylpropanoyl chloride was usedwithout any further purification, and suspended in 13 ml anhydrousacetonitrile. Cytidine (0.608 g, 2.5×10⁻³ mol) was added, and thereaction mixture was stirred at 60° C. for 24 h. The solvent wasevaporated off, and the residue treated with ether. The crude productwas stirred in 40 ml pyridine-methanol 1:1 at 80° C. for 20 h,whereafter it was evaporated to dryness and the product purified on acolumn of silica gel. Homogenous fractions were recrystallized to give0.446 g (35%) of the title compound as a white solid (mp. 164-166° C.).

¹H NMR (DMSO-d₆, 300 MHz) δ: 7.71 (1H, d, H-6), 7.2(2H, br.d, NH₂),6.1(1H, d, H-5), 5.88(1H, d, H-1′), 5.81(1H, d, OH-2′), 5.45(2H, m,CH═CH), 5.18(1H, m, OH-5′), 5.06(1H, dd, H-3′), 4.18(1H, m, H-2′),4.01(1H, m, H-4′), 3.75(2H, m, H-5′), 2.47(2H, t, CH₂—COO), 2.06(4H, m,CH₂—C═), 1.65(2H, m, CH₂—C—COO), 1.35(20H, m, CH₂), 0.97(3H, t, CH₃).

¹³C NMR (DMSO-d₆, 75 MHz) δ: 172.15(COO), 165.67(C4-N), 154.95(C═O),142.72(C-6), 130.11 and 130.08(CH═CH), 92.59(C-5), 86.24(C-1′),82.75(C-4′), 78.72(C-3′), 72.29(C-2′), 61.15(C-5′), 33.43, 31.97, 31.30,29.03, 28.99, 28.85, 28.73, 28.53, 28.41, 28.36, 24.40, 22.12(CH₂),13.97(CH₃).

EXAMPLE 3

5′-O-(cis-11-eicosenoyl) 1-β-D-arabinofuranosyl-cytosine

To a suspension of Ara-C-HCl (0.87 g, 3.1×10⁻³ mol) in 30 mlN,N-dimethylformamide was added a solution of cis-11-elcosenoyl chloride(1.06 g, 3.22×10⁻³ mol) in 30 ml DMA, and the reaction mixture wasstirred at 25° C. for 24 h. The solvents was evaporated at high vacuumand the residue was dissolved in 60 ml boiling ethanol to which wasadded 20 ml water and 20 ml saturated NaHCO₃ solution. The crude productwas filtered off at room temperature and dissolved in 100 ml boilingethanol (60% in water). The crude product was recrystallized fromethylacetate to give 1.1 g (66%) of the title compound as a white solid.

¹H NMR (DMSO-d₆, 300 MHz) δ: 7.45(1H, d, H-6), 7.1(2H, br.d, NH₂),6.08(1H, d, H-1′), 5.65(1H, d, H-5), 5.55(2H, m, OH-2′ and OH-3′),5.32(2H, m, OH═CH), 4.25(1H, m, H-5′), 4.15(1H, m, H-5′), 4.0-3.85(3H,m, H-2′, H-3′, H-4′), 2.33(2H, t, CH₂—COO), 1.95(4H, m, CH₂—C═), 1.5(2H,m, CH₂—C—COO), 1.25(24H, m, CH,₂), 0.85(3H, t, CH₃).

¹³C NMR (DMSO-d₆, 75 MHz) δ: 172.79(COO), 165.59(C-4), 155.08(C═O 2),142.78(C6), 129.60(CH═CH), 92.52(C-5), 86.21(C;1′), 81.82(C4′),76.75(C-3′), 74.25(C-2′), 63.76(C-5′), 33.41, 31.30, 29.11, 28.85,28.72, 28.60, 28.42, 26.57, 24.46, 22.11 (CH₂), 13.94(CH₃).

Ref.:

1. D. T. Gish et al.; J. Med. Chem. 14 (1971) 1159

2. E. K. Hamamura et al., J. Med. Chem. 19 (1976) 667

3. E. K. Hamamura et al, J. Med. Chem. 19 (1976) 654

What is claimed is:
 1. An Ara-C derivative of formula (I):

wherein R is selected from the group consisting of elaidoyl,cis-eicosenoyl and trans-eicosenoyl.
 2. A pharmaceutical composition,comprising an Ara-C derivative of formula (I), as defined in claim 1,and a pharmaceutically acceptable carrier or excipient therefor.
 3. AnAra-C derivative of formula (I):

wherein R is eicosanoyl.
 4. A pharmaceutical composition, comprising anAra-C derivative of formula (I), as defined in claim 3, and apharmaceutically acceptable carrier or excipient therefor. 5.5′-elaidoyl-Ara-C.
 6. A pharmaceutical composition, comprising5′-elaidoyl-Ara-C and a pharmaceutically acceptable carrier or excipienttherefor.
 7. 5′-cis-eicosenoyl-Ara-C.
 8. A pharmaceutical composition,comprising 5′-cis-eicosenoyl-Ara-C and a pharmaceutically acceptablecarrier or excipient therefor.
 9. 5′-trans-eicosenoyl-Ara-C.
 10. Apharmaceutical composition, comprising 5′-trans-eicosenoyl-Ara-C and apharmaceutically acceptable carrier or excipient therefor.